Effect of TRIP12, UBR5, or HOIP in gene expression

The protein ubiquitylation is under the equilibrium between ubiquitin conjugation and deconjugation. How substrates stabilized by deubiquitylation are directed for degradation remains unclear. Branched ubiquitin chains promote substrate degradation through the proteasome, but the underlying mechanisms are not fully understood. TRIP12 and UBR5 are HECT-type E3s specific for the K29 and K48 linkages, respectively. Here, we show that the deubiquitylase (DUB) OTUD5 is cooperatively modified by TRIP12 and UBR5, resulting in the conjugation of K29/K48 branched ubiquitin chains and accelerated proteasomal degradation. The TRIP12–OTUD5 antagonism regulates TNF-–induced NF-B signaling. Mechanistically, although OTUD5 readily cleaves K48 linkages, K29 linkages are resistant against OTUD5 activity. Consequently, K29 linkages overcome OTUD5 DUB activity to facilitate UBR5-dependent K48-linked chain branching. This mechanism is applicable to other TRIP12 substrates associated with OTUD5. These results reveal a unique cellular strategy in which the combination of DUB-resistant and proteasome-targeting ubiquitin linkages efficiently promotes the degradation of substrates protected by deubiquitylation, underscoring the role of branched ubiquitin chains in shifting the ubiquitin conjugation/deconjugation equilibrium. HT1080 cells were transfected with the siRNAs for TRIP12, UBR5, or HOIP. The cells were lysed 72h after the transfection, and total RNA was isolated.